为梭状体开发拉瓦尔斑马鱼感染模型
1Division of Cellular and Molecular Neurobiology, Zoological Institute,Technische Universität Braunschweig, 2Department of Molecular Biotechnology,Turkish-German University, 3Institut für Mikrobiologie,Technische Universität Braunschweig, 4Braunschweig Integrated Centre of Systems Biology, 5Helmholtz Centre for Infection Research
Summary
这里介绍的是一种安全有效的方法，通过微注射和非侵入性微藻感染斑马鱼幼虫荧光标记厌氧C.困难。
Abstract
梭状体困难感染 （CDI） 被认为是美国最常见的医疗保健相关胃肠道感染之一。已描述对C.困难的先天免疫反应，但中性粒细胞和巨噬细胞在CDI中的确切作用较少。在目前的研究中，达尼奥雷里奥（斑马鱼）幼虫被用来建立一个C.困难感染模型，用于成像这些先天免疫细胞在体内的行为和合作。为了监测梭菌，已经建立了一个使用荧光染料的标签协议。局部感染是通过标记C.困难的显微注射实现的，这种注射在斑马鱼肠道中积极生长，并模仿CDI中的肠道上皮损伤。然而，这种直接感染方案是侵入性的，导致显微伤口，这可能会影响实验结果。因此，这里描述了一种更无创的微藻协议。该方法包括通过张口插管将C.困难细胞直接输送到斑马鱼幼虫的肠道。这种感染方法与C.困难器的自然感染途径相近。
Introduction
梭菌是一种克阳性、孢子成形、厌氧和毒素产生的杆菌，是胃肠道1严重感染的主要原因。CDI的典型症状包括腹泻，腹痛，和致命的假膜结肠炎，它有时可能导致死亡1，2。有证据表明，宿主免疫反应在这种疾病的进展和结果中都起着关键作用。除了免疫反应，本地肠道微生物群对CDI4的发病和发病机制至关重要。在过去十年中，由于出现C.困难性超毒菌株（BI/NAP1/027）5、6 ，CDI的病例数和死亡率均显著增加。更好地了解潜在的免疫机制和在CDI期间微生物群的作用将有助于导致新的治疗发展和进步，从而更好地控制这一流行病。
一些动物模型，如仓鼠和老鼠，已经开发出来，以提供洞察对C.困难7，8的免疫防御。然而，对先天免疫细胞的作用仍然知之甚少，特别是因为先天免疫细胞的行为主要来自组织学分析或体外培养细胞。因此，建立一个透明的斑马鱼模型，揭示活体脊椎动物有机体内部对C.困难的先天免疫反应，将有助于此类研究。斑马鱼幼虫具有功能性先天免疫系统，但它们缺乏适应性免疫系统，直到受精9周后4-6周。这一独特特征使斑马鱼幼虫成为研究CDI中先天免疫细胞的分离反应和功能的优秀模型。
本报告描述了使用斑马鱼幼虫研究梭菌和先天免疫细胞（如巨噬细胞和嗜中性粒细胞）之间的相互作用的新方法。首先，提出了一种局部的显微注射方案，包括C.困难接种和染色。利用体内共聚焦延移成像，记录中性粒细胞和巨噬细胞对感染部位的反应，并观察嗜中性粒细胞和巨噬细胞对细菌的噬菌体。然而，据报道，注射本身导致组织损伤，并导致独立于细菌10的白细胞的招募。因此，随后描述了一种非侵入性微藻方案，用于将C.困难输送到斑马鱼幼虫的肠道。先前的研究已经表明，本地胃肠道微生物群保护宿主免受C.困难11的殖民化。因此，诺生物斑马鱼幼虫也被用来使被感染的斑马鱼容易感染。之后，进行肠道解剖以恢复可行的C.困难，并验证它们在斑马鱼肠道中的持续时间。
Protocol
此处描述的所有动物工作均按照法律规定（欧盟指令 2010/63、许可证 AZ 325.1.53/56.1-TUBS 和许可证 AZ 33.9-42502-04-14/1418）进行。 1. 低熔甘蔗、凝胶板和微注射/微加注针的制备 在 10 mL 的 30% Daniau 的介质（0.12 mM MgSO4， 0.18 mM Ca [NO3+2]） 0.21 mM KCl） 中溶解 0.08 g 低熔融甘蔗 （材料表， 甘蔗 A2576）， 1.5 mM HEPES （pH = 7.2） 和 17.4 mM NaCl，在室?…
Representative Results
梭子是严格无氧的，但荧光蛋白的色度通常需要氧气才能成熟。为了克服这个问题，荧光染料被用来染色活的C.困难细胞，这些细胞正在积极生长（R20291，核糖核酸型027菌株;图 1A.使用Gal4/UAS系统，生成稳定的转基因斑马鱼线进行活成像，其中mpeg1.1或lyZ启动子以Gal4依赖性的方式驱动巨噬细胞和中性粒细胞中EGFP荧光蛋白的表达。 <p cla…
Discussion
提出的方法修改和扩展了现有的方法，通过执行注射和微藻10，14感染斑马鱼幼虫。它还演示了一种研究斑马鱼幼虫22厌氧病原体的方法。此外，该议定书还有助于分析CDI和斑马鱼中C.困难的体内先天免疫细胞反应。该方法可重复，易于在常规实验室或临床环境中进行。
监测白细胞的C.困难的噬菌体， ?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢蒂莫·弗里奇对动物的精心照料。我们感谢科斯特和施泰纳特实验室的成员的支持和有益的讨论。我们感谢韩丹丹博士对手稿的批判性阅读。我们感谢下萨克森州联邦州尼德塞奇斯·沃拉布（VWZN2889）的资助。
Materials
| Agarose | Sigma-Aldrich | A2576 | Ultra-low gelling agarose |
| Agarose low-melting (LM) | Pronadisa | 8050 | It is used in agarose plates |
| BacLight Red Bacterial Stain | Thermo Fisher Scientific | B35001 | Fluorescent dye |
| Brain-Heart-Infusion Broth | Carl Roth GmbH | X916.1 | |
| Brass (wild-type) | deficient in melanin synthesis, used to generate stable transgenic lines | ||
| Calcium nitrate (Ca(NO3)2) | Sigma-Aldrich | C1396 | |
| Capillary Glass | Harvard Apparatus | 30-0019 | Injection needles |
| Clostridioides difficile | R20291,, a ribotype 027 strain, TcdA+/TcdB+/CDT+ production | ||
| DMSO | Carl Roth GmbH | A994 | |
| FIJI | open-source platform | Image processing | |
| HEPES | Carl Roth GmbH | 6763 | |
| Horizontal needle puller | Sutter instrument Inc | P-87 | |
| L-cysteine | Sigma-Aldrich | 168149 | |
| Leica Application Suite X (LAS X) | Leica | Image processing | |
| Magnesium sulfate (MgSO4) | Carl Roth GmbH | P026 | |
| Micro injector | eppendorf | 5253000017 | |
| Microinjection molds | Adaptive Science Tools | TU1 | |
| Leica SP8 confocal microscope | Leica | ||
| Phenol Red | Sigma-Aldrich | P0290 | |
| Potassium chloride (KCl) | Carl Roth GmbH | 5346 | |
| Sodium chloride (NaCl) | Carl Roth GmbH | 9265 | |
| Taurocholate | Carl Roth GmbH | 8149 | |
| Tg(lyZ: KalTA4)bz17/Tg(4xUAS-E1b:EGFP)hzm3 | stable transgenic line in which in which the lyZ promoters drive the expression of EGFP fluorescent protein in neutrophils | ||
| Tg(mpeg1.1: KalTA4)bz16/Tg(4xUAS-E1b:EGFP)hzm3 | stable transgenic line in which in which the mpeg1.1 drive the expression of EGFP fluorescent protein in macrophages | ||
| Tricaine | Sigma-Aldrich | E10521 | |
| Yeast extract | BD Bacto | 212750 |
Referências
- Rupnik, M., Wilcox, M. H., Gerding, D. N. Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nature Reviews Microbiology. 7, 526-536 (2009).
- Yang, Z., et al. Mechanisms of protection against Clostridium difficile infection by the monoclonal antitoxin antibodies actoxumab and bezlotoxumab. Infection and Immunity. 83, 822-831 (2015).
- Kelly, C. P., Kyne, L. The host immune response to Clostridium difficile. Journal of Medical Microbiology. 60, 1070-1079 (2011).
- Britton, R. A., Young, V. B. Interaction between the intestinal microbiota and host in Clostridium difficile colonization resistance. Trends in Microbiology. 20, 313-319 (2012).
- Goorhuis, A., et al. Emergence of Clostridium difficile Infection Due to a New Hypervirulent Strain, Polymerase Chain Reaction Ribotype 078. Clinical Infectious Diseases. 47, 1162-1170 (2008).
- Pépin, J., et al. Emergence of fluoroquinolones as the predominant risk factor for Clostridium difficile-associated diarrhea: a cohort study during an epidemic in Quebec. Clinical Infectious Diseases. 41, 1254-1260 (2005).
- Merrigan, M. M., Sambol, S. P., Johnson, S., Gerding, D. N. Prevention of Fatal Clostridium difficile -Associated Disease during Continuous Administration of Clindamycin in Hamsters. The Journal of Infectious Diseases. 188, 1922-1927 (2003).
- Chen, X., et al. A Mouse Model of Clostridium difficile-Associated Disease. Gastroenterology. 135, 1984-1992 (2008).
- Page, D. M., et al. An evolutionarily conserved program of B-cell development and activation in zebrafish. Blood. 122, 1-12 (2014).
- Benard, E. L., et al. Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens. Journal of Visualized Experiments. (61), e3781 (2012).
- Theriot, C. M., Young, V. B. Interactions Between the Gastrointestinal Microbiome and Clostridium difficile. Annual Review of Microbiology. 69, 445-461 (2015).
- Pham, L. N., Kanther, M., Semova, I., Rawls, J. F. Methods for generating and colonizing gnotobiotic zebrafish. Nature Protocols. 3, 1862-1875 (2008).
- Ransom, E. M., Ellermeier, C. D., Weiss, D. S. Use of mCherry red fluorescent protein for studies of protein localization and gene expression in Clostridium difficile. Applied and Environmental Microbiology. 81 (5), 1652-1660 (2015).
- Cocchiaro, J. L., Rawls, J. F. Microgavage of Zebrafish Larvae. Journal of Visualized Experiments. (72), e4434 (2013).
- Chen, X., et al. A Mouse Model of Clostridium difficile-Associated Disease. Gastroenterology. 135 (6), 1984-1992 (2008).
- Hutton, M. L., Mackin, K. E., Chakravorty, A., Lyras, D. Small animal models for the study of Clostridium difficile disease pathogenesis. FEMS Microbiology Letters. 352, 140-149 (2014).
- Brugman, S. The zebrafish as a model to study intestinal inflammation. Developmental & Comparative Immunology. 64, 82-92 (2016).
- Goulding, D., et al. Distinctive profiles of infection and pathology in hamsters infected with Clostridium difficile strains 630 and B1. Infection and Immunity. 77, 5478-5485 (2009).
- Toh, M. C., et al. Colonizing the Embryonic Zebrafish Gut with Anaerobic Bacteria Derived from the Human Gastrointestinal Tract. Zebrafish. 10, 194-198 (2013).
- Bloemberg, G. V., et al. Comparison of static immersion and intravenous injection systems for exposure of zebrafish embryos to the natural pathogen Edwardsiella tarda. BMC Immunology. 12, 58 (2011).
- Díaz-Pascual, F., Ortíz-Severín, J., Varas, M. A., Allende, M. L., Chávez, F. P. In vivo host-pathogen interaction as revealed by global proteomic profiling of zebrafish larvae. Frontiers in Cellular and Infection Microbiology. 7, 1-11 (2017).
- Valenzuela, M. J., et al. Evaluating the capacity of human gut microorganisms to colonize the zebrafish larvae (Danio rerio). Frontiers in Microbiology. 9, (2018).
Citar este artigo
Li, J., Ünal, C. M., Namikawa, K., Steinert, M., Köster, R. W. Development of a Larval Zebrafish Infection Model for Clostridioides difficile. J. Vis. Exp. (156), e60793, doi:10.3791/60793 (2020).